RRC ID 52860
Author Lin MT, Fukazawa R, Miyajima-Nakano Y, Matsushita S, Choi SK, Iwasaki T, Gennis RB.
Title Escherichia coli auxotroph host strains for amino acid-selective isotope labeling of recombinant proteins.
Journal Methods Enzymol
Abstract Enrichment of proteins with isotopes such as (2)H, (15)N, and (13)C is commonly carried out in magnetic resonance and vibrational spectroscopic characterization of protein structures, mechanisms, and dynamics. Although uniform isotopic labeling of proteins is straightforward, efficient labeling of proteins with only a selected set of amino acid types is often challenging. A number of approaches have been described in the literature for amino acid-selective isotope labeling of proteins, each with its own limitations. Since Escherichia coli represents the most cost-effective and widely used host for heterologous production of foreign proteins, an efficient method to express proteins selectively labeled with isotopes would be highly valuable for these studies. However, an obvious drawback is misincorporation and dilution of input isotope labels to unwanted amino acid types due to metabolic scrambling in vivo. To overcome this problem, we have generated E. coli auxotroph strains that are compatible with the widely used T7 RNA polymerase overexpression systems and that minimize metabolic scrambling. We present several examples of selective amino acid isotope labeling of simple and complex proteins with bound cofactors, as an initial guide for practical applications of these E. coli strains.
Volume 565
Pages 45-66
Published 2015-1-1
DOI 10.1016/bs.mie.2015.05.012
PII S0076-6879(15)00310-9
PMID 26577727
MeSH Amino Acids / chemistry* Escherichia coli / chemistry* Escherichia coli / classification Escherichia coli / genetics Escherichia coli Proteins / chemistry* Isotope Labeling* Recombinant Proteins / chemistry Species Specificity
IF 1.394
Times Cited 9
DNA material RF1 (RDB15945) RF2 (RDB15946) RF4 (RDB15947) RF5 (RDB15948) RF6 (RDB15949) RF8 (RDB15950) RF10 (RDB15951) RF11 (RDB15952) RF12 (RDB15953) RF13 (RDB15954).